The Polymerase Chain Reaction (PCR) amplifies DNA sequences through repeated cycles of template denaturation, primer annealing and elongation. Reverse transcription (RT) coupled with PCR (RT-PCR) combines cDNA synthesis from mRNA templates with PCR amplification to provide a rapid and sensitive method for detection, conversion and recovery of mRNA as DNA. The RT-PCR process can be performed in either one-tube or two-tube formats. In one-tube RT-PCR, the RT and PCR take place successively in a single tube using a mutual buffer for both reactions. In two-tube RT-PCR, RT and PCR are carried out separately. It has been shown that one-tube RT-PCR has the greater sensitivity and that as little as 100 copies of mRNA can be amplified (#TB220, Promega). In two tube RT-PCR, each step can be optimised separately and it may produce higher yields of DNA in some circumstances (TechNotes 9[6], Ambion).
High sensitivity (i.e. obtaining sufficient amount of DNA from as little template as possible) and high specificity (i.e. amplifying only the desired template) are key to successful PCR. They are affected by many factors including the choice of appropriate DNA polymerases, design of suitable primers, suitable buffers, thermal cycling parameters and also the quality of templates.
A single primer PCR approach has been developed for cloning unknown DNA sequences (Hermann et al., (2000) BioTechniques 29: 1176-1180) and elimination of primer-dimer accumulation in PCR (Brownie et al., (1997) Nucleic Acids Res. 25: 3235-3241). The single primer PCR uses one primer in the PCR mixture to amplify DNA having identical flanking sequences at both ends. Recently, this approach has been used to amplify single molecules of double-stranded DNA through 80 PCR cycles, a method termed single-molecule PCR (SM-PCR) (Rungpragayphan et al., (2002) J. Mol. Biol. 318: 395-405). In this method, double-stranded DNA is first amplified by PCR using two primers to introduce a tag sequence at both ends, after which the modified DNA is diluted and used as the template for SM-PCR (Rungpragayphan et al., (2002) J. Mol. Biol. 318: 395-405). However, none of these methods provided a sensitive procedure for cDNA recovery from mRNA
A number of display technologies have been developed for selection of proteins. Using the principle of coupling phenotype (protein) to genotype (gene), proteins have been successfully displayed on phage, cell surface and virus or ribosome, plasmid and mRNA. Prokaryotic and eukaryotic ribosome display systems have been used for selection of peptides, single-chain antibodies, enzymes, stable protein scaffolds and other ligand-binding domains. Display technology recovers DNA through the functionality of the encoded protein. A review of ribosome display technology is provided by He & Taussig (2002) Briefings in Functional Genomics and Proteomics, 1(2): 204-212.
Sensitive DNA recovery from mRNA is required in ribosome display. This cell-free protein display method allows the selection and evolution of proteins in vitro (He and Taussig (1997) Nucleic Acids Res. 25: 5153-5134; Hanes and Pluckthun, (1997) Proc. Natl. Acad. Sci. USA 94: 4937-4942). The method generates a library of ribosome display complexes, which are protein-ribosome-mRNA (PRM) complexes, from a diversity of DNA molecules by cell free expression, followed by capture of specific PRM complexes with a ligand through binding interaction of the displayed nascent protein. The associated mRNA is then retrieved and amplified as cDNA by RT-PCR. A key step in ribosome display is the efficient recovery of genetic material from PRM complexes after selection. A highly sensitive recovery method would allow rare species to be isolated from very large libraries. Currently, two principal recovery methods are employed. One is a ribosome disruption procedure used in prokaryotic ribosome display, which releases mRNA by the dissociation of ribosome complexes with EDTA followed by RT-PCR (Hanes and Pluckthun, 1997). The other method is an in situ RT-PCR method used in eukaryotic ribosome display (He and Taussig, 1997), which recovers DNA directly from PRM complexes without ribosome disruption through the use of a primer hybridising at least 60 nucleotides upstream of the 3′ end of the mRNA in order to avoid the region occupied by stalling ribosome. It has been demonstrated that the in situ RT-PCR procedure is more effective for recovery of DNA from eukaryotic ribosome complexes than the prokaryotic ribosome disruption method (He and Taussig, 2002 Briefings Func Genomics & Proteomics 1: 204-212). However, both methods require a sensitive procedure to recover cDNA from mRNA.
Sensitive recovery of low levels of mRNA would also be extremely useful in techniques such as single cell RT-PCR where for example, gene expression patterns are to be studied. Many cellular genes are expressed at very low levels. These are hard or even impossible to recover by traditional RT-PCR methods. Currently, repeated rounds of PCR reactions are usually performed (Gaynor et al., (1996) Biotechniques 21: 286-291). This can lead to artificial errors and meticulous controls are required. The RT-PCR method described here is sensitive enough such that only a single PCR reaction procedure is required, even when only a single cDNA molecule or a small number of DNA molecules is present as the initial PCR template.